Staphylococcus aureus is capable of infecting nearly every vertebrate organ system, triggering the formation of characteristic tissue lesions known as abscesses. Understanding how S. aureus survives within abscesses is critical to the development of new therapeutics, as these lesions represent the pathogen niche during infection. In this application, we will leverage a powerful mass spectrometry-based imaging platform to identify host and bacterial factors that contribute to staphylococcal disease. By defining how tissue niche and host biology drive molecular heterogeneity in abscesses, we will uncover new targets for tailored anti-staphylococcal therapeutics. Historically, it has been technically challenging to study the bacterial and host factors that contribute to abscess physiology for two primary reasons. First, approaches that seek to preserve abscess architecture within a tissue sample are inherently limited to the study of known microbial and host targets. This applies to techniques such as immunohistochemistry and fluorescence in situ hybridization, which can define the spatial distribution of analytes in a tissue but rely on pre-existing knowledge of targets. These approaches, by definition, cannot be used to discover unknown bacterial or host factors that contribute to disease. Conversely, discovery-based methods such as RNA sequencing or proteomics can identify novel, disease-associated analytes, but require destructive tissue processing that eliminates information regarding the spatial orientation of microbial and host molecules. To overcome these technical limitations, we created a mass spectrometry-based imaging platform to identify host and microbial analytes in abscessed tissue during invasive S. aureus infection. Because imaging mass spectrometry (IMS) does not require probes or detection reagents, this platform can define the localization and abundance of abscess-associated analytes in a spatially-defined manner, thereby enabling the discovery of microbial and host factors that contribute to disease pathogenesis. When applied to a model of disseminated S. aureus infection, this IMS-based platform enabled three-dimensional molecular imaging of the staphylococcal- host interface and powered the discovery of bacterial proteins that mark the pathogen niche within abscesses. Although individual abscesses typically have a similar histologic appearance, our IMS-based analysis revealed significant molecular heterogeneity between S. aureus lesions. We hypothesize that abscesses display molecular heterogeneity in response to tissue niche, antibacterial immune responses, and comorbid host conditions. To test this hypothesis, we will couple our IMS platform with laser capture microdissection to enable spatially-resolved proteomics of the staphylococcal-host interface. The proposed Aims will define the molecular architecture of abscesses across S. aureus infected tissues and determine how innate immune effector cells and host comorbidities drive heterogeneity in bacterial physiology and abscess molecular architecture in situ. In total, these experiments will decipher how host biology drives molecular heterogeneity during invasive infection.